UV excitable energy transfer reagents

ABSTRACT

Novel energy transfer dyes which can be used with shorter wavelength light sources are provided. These dyes include a donor dye with an absorption maxima at a wavelength between about 250 to 450 nm and an acceptor dye which is capable of absorbing energy emitted from the donor dye. One of the energy transfer dyes has a donor dye which is a member of a class of dyes having a coumarin or pyrene ring structure and an acceptor dye which is capable of absorbing energy emitted from the donor dye, wherein the donor dye has an absorption maxima between about 250 and 450 nm and the acceptor dye has an emission maxima at a wavelength greater than about 500 nm.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to fluorescent dyes and, morespecifically, energy transfer fluorescent dyes and their use.

[0003] 2. Description of Related Art

[0004] A variety of fluorescent dyes have been developed for labelingand detecting components in a sample. In general, fluorescent dyespreferably have a high quantum yield and a large extinction coefficientso that the dye may be used to detect small quantities of the componentbeing detected. Fluorescent dyes also preferably have a large Stokes'shift (i.e., the difference between the wavelength at which the dye hasmaximum absorbance and the wavelength at which the dye has maximumemission) so that the fluorescent emission is readily distinguished fromthe light source used to excite the dye.

[0005] One class of fluorescent dyes which has been developed is energytransfer fluorescent dyes. In general, energy transfer fluorescent dyesinclude a donor fluorophore and an acceptor fluorophore. In these dyes,when the donor and acceptor fluorophores are positioned in proximitywith each other and with the proper orientation relative to each other,the energy emission from the donor fluorophore is absorbed by theacceptor fluorophore and causes the acceptor fluorophore to fluoresce.It is therefore important that the excited donor fluorophore be able toefficiently absorb the excitation energy of the donor fluorophore andefficiently transfer the energy to the acceptor fluorophore.

[0006] A variety of energy transfer fluorescent dyes have been describedin the literature. For example, U.S. Pat. No. 4,996,143 and WO 95/21266describe energy transfer fluorescent dyes where the donor and acceptorfluorophores are linked by an oligonucleotide chain. Lee, et al.,Nucleic Acids Research 20:10 2471-2483 (1992) describes an energytransfer fluorescent dye which includes 5-carboxy rhodamine linked to4′-aminomethyl-5-carboxy fluorescein by the 4′-aminomethyl substituenton fluorescein. U.S. Pat. No. 5,847,162 describes additional classes ofenergy transfer dyes.

[0007] Several diagnostic and analytical assays have been developedwhich involve the detection of multiple components in a sample usingfluorescent dyes, e.g. flow cytometry (Lanier, et al., J. Immunol. 132151-156 (1984)); chromosome analysis (Gray, et al., Chromosoma 73 9-27(1979)); and DNA sequencing. For these assays, it is desirable tosimultaneously employ a set of two or more spectrally resolvablefluorescent dyes so that more than one target substance can be detectedin the sample at the same time. Simultaneous detection of multiplecomponents in a sample using multiple dyes reduces the time required toserially detect individual components in a sample. In the case ofmulti-loci DNA probe assays, the use of multiple spectrally resolvablefluorescent dyes reduces the number of reaction tubes that are needed,thereby simplifying the experimental protocols and facilitating themanufacturing of application-specific kits. In the case of automated DNAsequencing, the use of multiple spectrally resolvable fluorescent dyesallows for the analysis of all four bases in a single lane therebyincreasing throughput over single-color methods and eliminatinguncertainties associated with inter-lane electrophoretic mobilityvariations. Connell, et al., Biotechniques 5 342-348 (1987); Prober, etal., Science 238 336-341 (1987), Smith, et al., Nature 321 674-679(1986); and Ansorge, et al., Nucleic Acids Research 15 4593-4602 (1989).

[0008] There are several difficulties associated with obtaining a set offluorescent dyes for simultaneously detecting multiple target substancesin a sample, particularly for analyses requiring an electrophoreticseparation and treatment with enzymes, e.g., DNA sequencing. Forexample, each dye in the set must be spectrally resolvable from theother dyes. It is difficult to find a collection of dyes whose emissionspectra are spectrally resolved, since the typical emission bandhalf-width for organic fluorescent dyes is about 40-80 nanometers (nm)and the width of the available spectrum is limited by the excitationlight source. As used herein the term “spectral resolution” in referenceto a set of dyes means that the fluorescent emission bands of the dyesare sufficiently distinct, i.e., sufficiently non-overlapping, thatreagents to which the respective dyes are attached, e.g.polynucleotides, can be distinguished on the basis of the fluorescentsignal generated by the respective dyes using standard photodetectionsystems, e.g. employing a system of band pass filters andphotomultiplier tubes, charged-coupled devices and spectrographs, or thelike, as exemplified by the systems described in U.S. Pat. Nos.4,230,558, 4,811,218, or in Wheeless et al, pgs. 21-76, in FlowCytometry: Instrumentation and Data Analysis (Academic Press, New York,1985).

[0009] The fluorescent signal of each of the dyes must also besufficiently strong so that each component can be detected withsufficient sensitivity. For example, in the case of DNA sequencing,increased sample loading can not compensate for low fluorescenceefficiencies, Pringle et al., DNA Core Facilities Newsletter, 1 15-21(1988). The fluorescent signal generated by a dye is generally greatestwhen the dye is excited at its absorbance maximum. It is thereforepreferred that each dye be excited at about its absorbance maximum.

[0010] A further difficulty associated with the use of a set of dyes isthat the dyes generally do not have the same absorbance maximum. When aset of dyes are used which do not have the same absorbance maximum, atrade off is created between the higher cost associated with providingmultiple light sources to excite each dye at its absorbance maximum, andthe lower sensitivity arising from each dye not being excited at itsabsorbance maximum.

[0011] In addition to the above difficulties, the charge, molecularsize, and conformation of the dyes must not adversely affect theelectrophoretic mobilities of the fragments. The fluorescent dyes mustalso be compatible with the chemistry used to create or manipulate thefragments, e.g., DNA synthesis solvents and reagents, buffers,polymerase enzymes, ligase enzymes, and the like.

[0012] Because of the multiple constraints on developing a set of dyesfor multicolor applications, particularly in the area of four color DNAsequencing, only a few sets of fluorescent dyes have been developed.Connell, et al., Biotechniques 5 342-348 (1987); Prober, et al., Science238 336-341 (1987); and Smith, et al., Nature 321 674-679 (1986); andU.S. Pat. No. 5,847,162.

[0013] Energy transfer fluorescent dyes possess several features whichmake them attractive for use in the simultaneous detection of multipletarget substances in a sample, such as in DNA sequencing. For example, asingle donor fluorophore can be used in a set of energy transferfluorescent dyes so that each dye has strong absorption at a commonwavelength. Then, by varying the acceptor fluorophore in the energytransfer dye, a series of energy transfer dyes having spectrallyresolvable fluorescence emissions can be generated.

[0014] Energy transfer fluorescent dyes also provide a larger effectiveStokes' shift than non-energy transfer fluorescent dyes. This is becausethe Stokes' shift for an energy transfer fluorescent dye is based on thedifference between the wavelength at which the donor fluorophoremaximally absorbs light and the wavelength at which the acceptorfluorophore maximally emits light. In general, a need exists forfluorescent dyes having larger Stokes' shifts.

[0015] The sensitivity of any assay using a fluorescent dye is dependenton the strength of the fluorescent signal generated by the fluorescentdye. A need therefore exists for fluorescent dyes which have a strongfluorescence signal. With regard to energy transfer fluorescent dyes,the fluorescence signal strength of these dyes is dependent on howefficiently the acceptor fluorophore absorbs the energy emission of thedonor fluorophore.

SUMMARY OF THE INVENTION

[0016] The present invention relates to energy transfer dyes which canbe used with shorter wavelength light sources. The present inventionalso relates to reagents which include the energy transfer dyes of thepresent invention. The present invention also relates to methods whichuse dyes and reagents adapted to shorter wavelength light sources. Kitsare also provided which include the dyes and reagents.

[0017] Energy transfer dyes are provided which include a donor dye withan absorption maxima at a wavelength between about 250 to 450 nm and anacceptor dye which is capable of absorbing energy from the donor dye.

[0018] It is noted that energy transfer may occur by a variety ofmechanisms. For example, the emission of the donor dye does not need tooverlap with the absorbance of the acceptor dye for many of the dyes ofthe present invention.

[0019] In one variation, the donor dye has an absorption maxima betweenabout 300 and 450 nm, more preferably between about 350 and 400 nm.

[0020] The acceptor dye preferably has an emission maxima greater thanabout 500 nm. In one variation, the acceptor dye has an emission maximaat a wavelength greater than about 550 nm. The acceptor dye may alsohave an emission maxima at a wavelength between about 500 and 700 nm.The acceptor dye may also be selected relative to the donor dye suchthat the acceptor dye has an emission maxima at a wavelength at leastabout 150 nm greater than the absorption maxima of the donor dye.

[0021] In another embodiment of the present invention, the energytransfer dye has a donor dye which is a member of a class of dyes havinga coumarin or pyrene ring structure and an acceptor dye which is capableof absorbing energy from the donor dye.

[0022] In one variation of this embodiment, the donor dye has anabsorption maxima between about 250 and 450 nm, preferably between about300 and 450 nm, and more preferably between about 350 and 400 nm.

[0023] In another variation of this embodiment, the acceptor dye has anemission maxima at a wavelength greater than about 500 nm, andoptionally more than 550 nm. The acceptor dye may also have an emissionmaxima at a wavelength between about 500 and 700 nm. The acceptor dyemay also be selected relative to the donor dye such that the acceptordye has an emission maxima at a wavelength at least about 150 nm greaterthan the absorption maxima of the donor dye.

[0024] An energy transfer dye according to the present invention mayalso have the structure of “antennae” dyes or dendrimers in which largenumbers of donor dyes are coupled to one acceptor dye where the donordye either has an absorption maxima between 250 and 450 nm or has acoumarin or pyrene ring structure.

[0025] The present invention also relates to fluorescent reagentscontaining any of the energy transfer dyes of the present invention. Ingeneral, these reagents include any molecule or material to which theenergy transfer dyes of the invention can be attached. The presence ofthe reagent is detected by the fluorescence of the energy transfer dye.One use of the reagents of the present invention is in nucleic acidsequencing.

[0026] Examples of classes of the fluorescent reagents includedeoxynucleosides and mono-, di- or triphosphates of a deoxynucleosidelabeled with an energy transfer dye. Examples of deoxynucleotidesinclude deoxycytosine, deoxyadenosine, deoxyguanosine or deoxythymidine,and analogs and derivatives thereof.

[0027] Other classes of the reagents include analogs and derivatives ofdeoxynucleotides which are not extended at the 3′ position by apolymerase. A variety of analogs and derivatives have been developedwhich include a moiety at the 3′ position to prevent extension includinghalides, acetyl, benzyl and azide groups. Dideoxynucleosides anddideoxynucleoside mono-, di- or triphosphates which cannot be extendedhave also been developed. Examples of dideoxynucleotides includedideoxycytosine, dideoxyadenosine, dideoxyguanosine or dideoxythymidine,and analogs and derivatives thereof.

[0028] The fluorescently labeled reagent may also be an oligonucleotide.The oligonucleotide may have a 3′ end which is extendable by using anucleotide polymerase. Such a labeled oligonucleotide may be used, forexample, as a dye-labeled primer in nucleic acid sequencing.

[0029] The present invention also relates to methods which use theenergy transfer dyes and reagents of the present invention. In oneembodiment, the method includes forming a series of different sizedoligonucleotides labeled with an energy transfer dye of the presentinvention, separating the series of labeled oligonucleotides based onsize and detecting the separated labeled oligonucleotides based on thefluorescence of the energy transfer dye.

[0030] In another embodiment, the method includes forming a mixture ofextended labeled primers by hybridizing a nucleic acid with anoligonucleotide primer in the presence of deoxynucleoside triphosphates,at least one dideoxynucleoside triphosphate and a DNA polymerase, theDNA polymerase extending the primer with the deoxynucleosidetriphosphates until a dideoxynucleoside triphosphate is incorporatedwhich terminates extension of the primer. Once terminated, the mixtureof extended primers are separated and the separated extended primersdetected by detecting an energy transfer dye of the present inventionthat was incorporated onto either the oligonucleotide primer, adeoxynucleotide triphosphate, or a dideoxynuceotide triphosphate.

[0031] The present invention also relates to methods for sequencing anucleic acid using the energy transfer dyes of the present invention. Inone embodiment, the method includes forming a mixture of extendedlabeled primers by hybridizing a nucleic acid sequence with anoligonucleotide primer in the presence of deoxynucleoside triphosphates,at least one dideoxynucleoside triphosphate and a DNA polymerase. Theoligonucleotide primer and/or the dideoxynucleotide is labeled with anenergy transfer dye of the present invention. The DNA polymerase is usedto extend the primer with the deoxynucleoside triphosphates until adideoxynucleoside triphosphate is incorporated which terminatesextension of the primer. The mixture of extended primers are thenseparated and the sequence of the nucleic acid determined by detectingthe energy transfer dye on the extended primer.

[0032] The present invention also relates to methods for detectingoligonucleotides and reagents labeled with energy transfer dyes usingshorter wavelength light sources. The light sources used in thesemethods preferably provide energy at a wavelength less than 450 nm. Inone variation, the light source provides energy at a wavelength betweenabout 250 and 450 nm, preferably between about 300 and 450 nm, and mostpreferably between about 350 and 450 nm. In one particular embodiment,the light source used provides energy at about 400 nm.

[0033] In one embodiment, the method includes forming a series ofdifferent sized oligonucleotides labeled with an energy transfer dye,separating the series of labeled oligonucleotides based on size anddetecting the separated labeled oligonucleotides based on thefluorescence of the energy transfer dye upon exposure to a shorterwavelength light source.

[0034] In another embodiment, the method includes forming a mixture ofextended labeled primers by hybridizing a nucleic acid with anoligonucleotide primer in the presence of deoxynucleoside triphosphates,at least one dideoxynucleoside triphosphate and a DNA polymerase, theDNA polymerase extending the primer with the deoxynucleosidetriphosphates until a dideoxynucleoside triphosphate is incorporatedwhich terminates extension of the primer. Once terminated, the mixtureof extended primers are separated. The separated extended primers aredetected by exposing the extended primer to light having a wavelengthbetween about 250 and 450 nm and measuring light emitted by an energytransfer dye at a wavelength greater than about 500 nm. The energytransfer dye is incorporated onto either the oligonucleotide primer, adeoxynucleotide triphosphate, or a dideoxynuceotide triphosphate.

[0035] The present invention also relates to methods for sequencing anucleic acid using a shorter wavelength light source. In one embodiment,the method includes forming a mixture of extended labeled primers byhybridizing a nucleic acid sequence with an oligonucleotide primer inthe presence of deoxynucleoside triphosphates, at least onedideoxynucleoside triphosphate and a DNA polymerase. The oligonucleotideprimer and/or the dideoxynucleotide is labeled with an energy transferdye adapted for use with a shorter wavelength light source. The DNApolymerase is used to extend the primer with the deoxynucleosidetriphosphates until a dideoxynucleoside triphosphate is incorporatedwhich terminates extension of the primer. The mixture of extendedprimers are then separated and the sequence of the nucleic aciddetermined by exposing the extended primer to light having a wavelengthbetween about 250 and 450 nm and measuring light emitted by the energytransfer dye at a wavelength greater than about 500 nm.

[0036] In a preferred variation of the embodiment, the extended primeris exposed to light having a wavelength between about 300 and 450 nm.The extended primer may also be exposed to light having a wavelengthbetween about 350 and 400 nm. In another preferred variation of theembodiment, the light emitted by the energy transfer dye has awavelength greater than about 550 nm. The light emitted by the energytransfer dye may also have a wavelength between about 500 and 700 nm. Inanother embodiment, the light emitted by the energy transfer dye has awave length at least about 150 nm greater than the wavelength of thelight to which the extended primer is exposed.

[0037] The present invention also relates to kits containing the dyesand reagents for performing DNA sequencing using the dyes and reagentsof the present invention. A kit may include a set of 2, 3, 4 or moreenergy transfer dyes or reagents of the present invention. Optionallythe kits may further include a nucleotide polymerase, additionalnucleotides and/or reagents useful for performing nucleic acidsequencing.

BRIEF DESCRIPTION OF THE FIGURES

[0038]FIG. 1 illustrates examples of energy transfer dyes according tothe present invention.

[0039]FIG. 2 illustrates examples of donor dyes which include a pyrenering structure.

[0040]FIG. 3 illustrates examples of donor dyes which include a coumarinring structure.

[0041]FIG. 4 illustrates the structure of a dendrimer energy-transferdye.

[0042]FIG. 5 illustrates classes of acceptor dyes including xanthenedyes, cyanine dyes, phthalocyanine dyes and squaraine dyes.

[0043]FIG. 6 illustrates the general structure of xanthene dyes andclasses of xanthene dyes like fluorescein, rhodamine and asymmetricbenzoxanthene.

[0044]FIG. 7 illustrates structures of acceptor dyes which may be usedin the dyes of the present invention.

[0045]FIG. 8 illustrates examples of —C(O)R₂₂— subunits of linkers whichmay be used in the present invention.

[0046]FIG. 9 illustrates the synthesis scheme of energy transfer dyeDYE104.

[0047]FIG. 10 illustrates the synthesis scheme of energy transfer dyeDYE106.

[0048]FIG. 11 illustrates the synthesis scheme of energy transfer dyeDYE108.

[0049]FIG. 12 shows the fluorescence emission spectra of energy transferdyes according to the present invention.

[0050]FIG. 13 illustrates the synthesis scheme of energy transfer dyeDYE120.

[0051]FIG. 14 shows the fluorescence emission spectra of energy transferdye DYE120 according to the present invention.

DETAILED DESCRIPTION

[0052] The present invention relates to energy transfer dyes which maybe used with shorter wavelength light sources. For example, the energytransfer dyes are preferably adapted to be excited at wavelengthsbetween about 250 and 450 nm. The present invention also relates toreagents which include the energy transfer dyes of the presentinvention. The present invention further relates to methods which usethe dyes and reagents. Kits are also provided which include the dyes andreagents.

[0053] I. Energy Transfer Dyes

[0054] The energy transfer dyes of the present invention include a donordye and an acceptor dye which is capable of emitting energy in responseto absorbing energy from the donor dye.

[0055] In one embodiment, the energy transfer dyes may be excited atwavelengths between about 250 and 450 nm. According to this embodiment,the donor dye preferably has an absorption maxima at a wavelengthbetween about 250 to 450 nm, more preferably between about 300 and 450nm, and most preferably between about 350 and 450 nm.

[0056] In another embodiment, the energy transfer dyes include an donordye having a coumarin or pyrene ring structure.

[0057] The acceptor dye may be any dye which is capable of absorbingenergy from the donor dye. In one embodiment, the acceptor dye has anemission maxima greater than about 500 nm, more preferably greater than550 nm. In another embodiment, the acceptor dye has an emission maximabetween about 500 and 700 nm. In another embodiment, the acceptor dye isselected such that it has an emission maxima at a wavelength at leastabout 150 nm greater than the absorption maxima of the donor dye.

[0058] The energy transfer dyes may also include a linker which couplesthe donor dye to the acceptor dye. The linker preferably couples thedonor dye to the acceptor dye such that the acceptor dye is able toabsorb substantially all of the energy by the donor dye.

[0059] Particular examples of energy transfer dyes of the presentinvention are illustrated in FIG. 1. In these examples5-carboxyfluorescein, which has an emission maxima of 523 nm, is used asthe acceptor dye. Coumarin-based donor dyes DYE116, which has anabsorption maxima at 376 nm, DYE114 (absorption maximum=328 nm), andDYE112 (absorption maximum=362 nm) or pyrene-based donor dye DYE110(absorption maximum=396 nm) are conjugated to a 5-carboxyfluoresceinacceptor derivatized with a 4-aminomethylbenzoic linker (5CF-B). Thestructures of the 5CF-B conjugates, DYE102, DYE104, DYE106, and DYE108,are shown in FIG. 1.

[0060] A. Donor Dye

[0061] In one embodiment, the donor dye has an absorption maxima at awavelength between about 250 to 450 nm, more preferably between about300 and 450 nm, most preferably between about 350 and 400 nm.

[0062] In another embodiment, the donor dye has a pyrene ring structure.As used herein, pyrene dyes include all molecules including the generalstructure

[0063] The present invention is intended to encompass all pyrene dyessince all may be used in the present invention. Particular examples ofpyrene dyes, DYE110, DYE122, DYE124 and DYE126, are illustrated in FIG.2. In the figure, X is a functional group which may be used to attachsubstituents, such as the acceptor dye, to the donor dye.

[0064] In another embodiment, the donor dye has a coumarin ringstructure. As used herein, coumarin dyes include all molecules includingthe general structure.

[0065] The present invention is intended to encompass all coumarin dyessince all may be used in the present invention. Particular examples ofcoumarin dyes are illustrated in FIG. 3. In the figure, X is afunctional group which may be used to attach substituents, such as theacceptor dye, to the donor dye.

[0066] The present invention also relates to energy transfer dyes wheremultiple donor dyes are coupled to an acceptor dye. Coumarin dyes arewater-soluble and coumarin conjugates show much better quantum yieldsthan larger dyes, for which the quantum yields in water are about ⅓ thatof free acceptor dyes. The present invention utilizes the small size andsolubility of the coumarins to synthesize “antennae” dyes or dendrimersin which large numbers of donor dyes are coupled to one acceptor dye. Anexample of a dendrimer energy transfer dye (DYE118) is shown in FIG. 4.

[0067] B. Acceptor Dye

[0068] The acceptor dye may be any dye which is capable of absorbingenergy from the donor dye. In one embodiment, the acceptor dye has anemission maxima greater than about 500 nm, more preferably greater than550 nm. In another embodiment, the acceptor dye has an emission maximabetween about 500 and 700 nm. In another embodiment, the acceptor dye isselected such that it has an emission maxima at a wavelength at leastabout 150 nm greater than the absorption maxima of the donor dye.

[0069] Examples of classes of acceptor dyes which may be used in theenergy transfer fluorescent dye of this embodiment include, but are notlimited to, xanthene dyes, cyanine dyes, phthalocyanine dyes andsquaraine dyes. The general structures of these dyes are illustrated inFIG. 5. The substituents illustrated on these dyes may be selected fromthe wide variety of substituents which may be incorporated onto thesedifferent classes of dyes since all dyes having the general xanthene,fluorescein, rhodamine, asymmetric benzoxanthene, cyanine,phthalocyanine and squaraine ring structures are intended to fall withinthe scope of this invention.

[0070] One particular class of acceptor dyes which may be used in theenergy transfer dyes of the present invention are xanthene dyes. As usedherein, xanthene dyes include all molecules having the general structureillustrated in FIG. 6 where Y₁ and Y₂ taken separately are eitherhydroxyl, oxygen, iminium or amine, the iminium and amine preferablybeing a tertiary iminium or amine. Examples of classes of xanthene dyesare fluorescein, rhodamine and asymmetric benzoxanthene classes of dyeswhich are also illustrated in FIG. 6. The substituents illustrated onthese dyes may be selected from the wide variety of substituents whichmay be incorporated onto these different classes of dyes since all dyeshaving the general xanthene, fluorescein, rhodamine, and asymmetricbenzoxanthene ring structures are intended to fall within the scope ofthis invention. Fluorescein and rhodamine dyes may be linked to asubstituent, such as an acceptor dye, a nucleoside, or anoligonucleotide, in a variety of locations. Illustrated with an asterik“*” in FIG. 6 are preferred locations for substitutions.

[0071] Fluorescein and rhodamine classes of dyes are members of aparticular subclass of xanthene dyes where R₁₇ is a phenyl orsubstituted phenyl having the general formula

[0072] Substituents X₁-X₅ on the phenyl ring can include hydrogen,fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne,sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacentsubstituents are taken together to form a ring, and combinationsthereof. As illustrated in FIG. 5, dyes where Y₁ is hydroxyl and Y₂ iscarboxyl are fluorescein dyes and where Y₁ is amine and Y₂ is iminiumare rhodamine dyes.

[0073] R₁₁-R₁₇ may be any substituent which is compatible with theenergy transfer dyes of the present invention, it being noted that theR₁₁-R₁₇ may be widely varied in order to alter the spectral and mobilityproperties of the dyes. Examples of R₁₁-R₁₇ substituents include, butnot limited to hydrogen, fluorine, chlorine, bromine, iodine, carboxyl,alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile,alkoxy, phenyl, substituted phenyl, where adjacent substituents aretaken together to form a ring, and combinations thereof.

[0074] In one embodiment, R₁₅ and R₁₆ are taken together to form asubstituted or unsubstituted benzene ring. This class of xanthene dyesare referred to herein as asymmetric benzoxanthene dyes and aredescribed in U.S. Pat. No. 5,840,999, entitled Asymmetric BenzoxantheneDyes, by Scott C. Benson, et al. which is incorporated herein byreference.

[0075] In one particular embodiment, the acceptor dye is a member of theclass of dyes where Y₁ is amine, Y₂ is iminium, and X₂ and X₅ arechlorine, referred to herein as 4,7-dichlororhodamine dyes. Dyes fallingwithin the 4,7-dichlororhodamine class of dyes and their synthesis aredescribed in U.S. Pat. No. 5,847,162, entitled: “4,7-DichlororhodamineDyes” which is incorporated herein by reference.

[0076] R₁₁-R₁₇ and X₁-X₅ may also each independently be a linking moietywhich may be used to attach the energy transfer dye to a reagent, suchas a nucleotide, nucleoside or oligonucleotide. Examples of linkingmoieties include isothiocyanate, sulfonyl chloride,4,6-dichlorotriazinylamine, succinimidyl ester, or other activecarboxylate whenever the complementary functionality is amine.Preferably the linking group is maleimide, halo acetyl, or iodoacetamidewhenever the complementary functionality is sulfhydryl. See R. Haugland,Molecular Probes Handbook of Fluorescent Probes and Research Chemicals,Molecular probes, Inc. (1992). In a particularly preferred embodiment,the linking group is an activated NHS ester formed from a carboxyl groupon either the donor or acceptor dye which can be reacted with anaminohexyl-oligomer to form a dye labeled oligonucleotide primer.

[0077] As used here, alkyl denotes straight-chain and branchedhydrocarbon moieties, i.e., methyl, ethyl, propyl, isopropyl,tert-butyl, isobutyl, sec-butyl, neopentyl, tert-pentyl, and the like.Substituted alkyl denotes an alkyl moiety substituted with any one of avariety of substituents, including, but not limited to hydroxy, amino,thio, cyano, nitro, sulfo, and the like. Haloalkyl denotes a substitutedalkyl with one or more halogen atom substituents, usually fluoro,chloro, bromo, or iodo. Alkene denotes a hydocarbon wherein one or moreof the carbon-carbon bonds are double bonds, and the non-double bondedcarbons are alkyl or substituted alkyl. Alkyne denotes a hydocarbonwhere one or more of the carbons are bonded with a triple bond and wherethe non-triple bonded carbons are alkyl or substituted alkyl moieties.Sulfonate refers to moieties including a sulfur atom bonded to 3 oxygenatoms, including mono- and di-salts thereof, e.g., sodium sulfonate,potassium sulfonate, disodium sulfonate, and the like. Amino refers tomoieties including a nitrogen atom bonded to 2 hydrogen atoms, alkylmoieties, or any combination thereof. Amido refers to moieties includinga carbon atom double bonded to an oxygen atom and single bonded to anamino moiety. Nitrile refers to moieties including a carbon atom triplebonded to a nitrogen atom. Alkoxy refers to a moiety including an alkylmoiety single bonded to an oxygen atom. Aryl refers to single ormultiple phenyl or substituted phenyl, e.g., benzene, naphthalene,anthracene, biphenyl, and the like.

[0078] In another embodiment, the acceptor dye is selected such that theacceptor dye has an emission maximum that is greater than about 500 nmand an emission maximum that is at least about 150 nm greater than theabsorption maxima of the donor dye. This class of dyes of the presentinvention exhibit unusually large Stokes' shifts, as measured by thedifference between the absorbance of the donor and the emission of theacceptor. In addition, these dyes exhibit efficient energy transfer inthat minimal donor fluorescence is observed. Interestingly, energy istransferred from the donor to the acceptor in some of the dyes belongingto this class even though the absorbance spectrum of the acceptor dyedoes not overlap with the emission spectrum of the donor dye.

[0079] Particular examples of acceptor dyes which may be used in thedyes of the present invention include, but are not limited to isomers ofcarboxyfluorescein (e.g., 5 and 6 carboxy), 4,7-dichlorofluoresceins,4,7-dichlororhodamines, fluoresceins, asymmetric benzoxanthene dyes,isomers of carboxy-HEX (e.g., 5 and 6 carboxy), NAN, CI-FLAN, TET, JOE,ZOE, rhodamine, isomers of carboxyrhodamine (e.g., 5 and 6 carboxy),isomers of carboxy R110 (e.g., 5 and 6 carboxy), isomers of carboxy R6G(e.g., 5 and 6 carboxy), 4,7-dichlorofluoresceins (See U.S. Pat. No.5,188,934), 4,7-dichlororhodamines (See U.S. Pat. No. 5,847,162),asymmetric benzoxanthene dyes (See U.S. Pat. No. 5,840,999), isomers ofN,N,N′,N′-tetramethyl carboxyrhodamine (TAMRA) (e.g., 5 and 6 carboxy),isomers of carboxy-X-rhodamine (ROX) (e.g., 5 and 6 carboxy) and Cy5.Illustrated in FIG. 7 are the structures of these dyes.

[0080] C. Linkers

[0081] The donor dye may be joined with the acceptor dye using a widevariety of linkers which have been developed, all of which are intendedto fall within the scope of the present invention. The energy transferdyes which include a linker may generally be illustrated as

[0082] DONOR - - - LINKER - - - ACCEPTOR

[0083] In a preferred embodiment, the linker joins the donor dye to theacceptor dye such that the acceptor dye absorbs substantially all of theenergy by the donor dye. While not being bound by theory, it is believedthat the efficiency of energy transmission from the donor dye to theacceptor dye is dependent upon the separation between the dyes andrelative orientation of the dyes. Described in U.S. Pat. No. 5,800,996are linkers which have been found to be effective for providing a veryhigh level of energy transfer between the donor and acceptor dye. U.S.Pat. No. 5,800,996 also describes methods for synthesizing dyesincorporating these linkers. U.S. Pat. No. 5,800,996 is incorporatedherein by reference in its entirety.

[0084] In one particular embodiment, the linker used in the energytransfer dyes of the present invention is such that the acceptor dyeabsorbs substantially all of the excitation energy by the donor dye.Such linkers may include a functional group which provides structuralrigidity to the linker. Examples of such functional groups include analkene, diene, alkyne, a five and six membered ring having at least oneunsaturated bond and/or having a fused ring structure.

[0085] Examples of functional groups with a five or six membered ringwith at least one unsaturatd bond and/or a fused ring structure includecyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan,thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran,pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine,indene, benzofuran, thionaphthene, indole and naphthalene.

[0086] One linker according to the present invention for linking a donordye to an acceptor dye in an energy transfer dye includes the subunitstructure —C(O)R₂₂—, where R₂₂ includes a functional group such as theones described above which provides structural rigidity. FIG. 8illustrates examples of —C(O)R₂₂— subunits of linkers which may be usedin the linkers of the present invention.

[0087] One embodiment of this linker has the general structure—R₂₁Z₁C(O)R₂₂R₂₈—, where R₂₁ is a C₁₋₅ alkyl attached to the donor dye,C(O) is a carbonyl group, Z₁ is either NH, sulfur or oxygen, R₂₂ is asubstituent which includes an alkene, diene, alkyne, a five and sixmembered ring having at least one unsaturated bond or a fused ringstructure which is attached to the carbonyl carbon, and R₂₈ includes afunctional group which attaches the linker to the acceptor dye.

[0088] In one embodiment of this linker, the linker has the generalstructure —R₂₁Z₁C(O)R₂₂R₂₉Z₂C(O)— where R₂₁ and R₂₂ are as detailedabove, Z₁ and Z₂ are each independently either NH, sulfur or oxygen, R₂₉is a C₁₋₅ alkyl, and the terminal carbonyl group is attached to the ringstructure of the acceptor dye. In the variation where Z₂ is nitrogen,the —C(O)R₂₂R₂₉Z₂— subunit forms an amino acid subunit.

[0089] A preferred embodiment of this linker is where R₂₁ and R₂₉ aremethylene, Z₁ and Z₂ are NH, and R₂₂ is benzene.

[0090] In yet another variation, the linker has the general formulaR₂₅Z₃C(O) or R₂₅Z₃C(O)R₂₆Z₄C(O) where R₂₅ is attached to the donor dye,C(O) is a carbonyl group and the terminal carbonyl group is attached tothe acceptor dye, R₂₅ and R₂₆ are each selected from the group of C₁₋₄alkyl, and Z₃ and Z₄ are each independently either NH, O or S.

[0091] In another variation of this embodiment, the linker includes aR₂₇Z₅C(O) group where R₂₇ is a C₁₋₅ alkyl attached to the donor dye, Z₅is either NH, sulfur or oxygen, and C(O) is a carbonyl group attached tothe acceptor dye.

[0092] II. Reagents Including Energy Transfer Dyes of the PresentInvention

[0093] The present invention also relates to reagents which incorporatean energy transfer dye according to the present invention. As describedin greater detail in Section III, these reagents may be used in a widevariety of methods for detecting the presence of a component in asample.

[0094] The reagents of the present invention include any molecule ormaterial to which the energy transfer dyes of the invention can beattached and used to detect the presence of the reagent based on thefluorescence of the energy transfer dye. Types of molecules andmaterials to which the dyes of the present invention may be attached toform a reagent include, but are not limited to proteins, polypeptides,polysaccharides, nucleotides, nucleosides, oligonucleotides,oligonucleotide analogs (such as a peptide nucleic acid), lipids, solidsupports, organic and inorganic polymers, and combinations andassemblages thereof, such as chromosomes, nuclei, living cells, such asbacteria, other microorganisms, mammalian cells, and tissues.

[0095] Preferred classes of reagents of the present invention arenucleotides, nucleosides, oligonucleotides and oligonucleotide analogswhich have been modified to include an energy transfer dye of theinvention. Examples of uses for nucleotide and nucleoside reagentsinclude, but are not limited to, labeling oligonucleotides formed byenzymatic synthesis, e.g., nucleoside triphosphates used in the contextof PCR amplification, Sanger-type nucleotide sequencing, andnick-translation reactions. Examples of uses for oligonucleotidereagents include, but are not limited to, as DNA sequencing primers, PCRprimers, oligonucleotide hybridization probes, and the like.

[0096] One particular embodiment of the reagents are labelednucleosides, such as cytosine, adenosine, guanosine, and thymidine,labeled with an energy transfer fluorescent dye of the presentinvention. These reagents may be used in a wide variety of methodsinvolving oligonucleotide synthesis. Another related embodiment arelabeled nucleotides (NTP), e.g., mono-, di- and triphosphate nucleosidephosphate esters. These reagents include, in particular, deoxynucleosidetriphosphates (dNTP), such as deoxycytosine triphosphate, deoxyadenosinetriphosphate, deoxyguanosine triphosphate, and deoxythymidinetriphosphate, labeled with an energy transfer fluorescent dye of thepresent invention. These reagents may be used, for example, aspolymerase substrates in the preparation of dye labeledoligonucleotides. These reagents also include labeled dideoxynucleosidetriphosphates (ddNTP), such as dideoxycytosine triphosphate,dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, anddideoxythymidine triphosphate, labeled with an energy transferfluorescent dye of the present invention. These reagents may be used,for example, in dye termination sequencing.

[0097] Another embodiment of reagents are oligonucleotides whichincludes an energy transfer dye of the present invention. These reagentsmay be used, for example, in dye primer sequencing.

[0098] As used herein, “nucleoside” refers to a compound consisting of apurine, deazapurine, or pyrimidine nucleoside base, e.g., adenine,guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, andthe like, linked to a pentose at the 1′ position, including 2′-deoxy and2′-hydroxyl forms, e.g. as described in Kornberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992). The term“nucleotide” as used herein refers to a phosphate ester of a nucleoside,e.g., mono, di and triphosphate esters, wherein the most common site ofesterification is the hydroxyl group attached to the C-5 position of thepentose. “Analogs” in reference to nucleosides include syntheticnucleosides having modified base moieties and/or modified sugarmoieties, e.g. described generally by Scheit, Nucleotide Analogs (JohnWiley, New York, 1980). The terms “labeled nucleoside” and “labelednucleotide” refer to nucleosides and nucleotides which are covalentlyattached to an energy transfer dye through a linkage.

[0099] As used herein, the term “oligonucleotide” refers to linearpolymers of natural or modified nucleoside monomers, including doubleand single stranded deoxyribonucleosides, ribonucleosides, α-anomericforms thereof, and the like. Usually the nucleoside monomers are linkedby phosphodiester linkages, where as used herein, the term“phosphodiester linkage” refers to phosphodiester bonds or analogsthereof including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like, including associatedcounterions, e.g., H, NH₄, Na, and the like if such counterions arepresent. The oligonucleotides range in size form a few monomeric units,e.g. 8-40, to several thousands of monomeric units. Whenever anoligonucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′→3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine,unless otherwise noted.

[0100] Nucleoside labeling can be accomplished using any of a largenumber of known nucleoside labeling techniques using known linkages,linking groups, and associated complementary functionalities. Thelinkage linking the dye and nucleoside should (i) be stable tooligonucleotide synthesis conditions, (ii) not interfere witholigonucleotide-target hybridization, (iii) be compatible with relevantenzymes, e.g., polymerases, ligases, and the like, and (iv) not quenchthe fluorescence of the dye.

[0101] Preferably, the dyes are covalently linked to the 5-carbon ofpyrimidine bases and to the 7-carbon of 7-deazapurine bases. Severalsuitable base labeling procedures have been reported that can be usedwith the invention, e.g. Gibson et al, Nucleic Acids Research, 156455-6467 (1987); Gebeyehu et al, Nucleic Acids Research, 15 4513-4535(1987); Haralambidis et al, Nucleic Acids Research, 15 4856-4876 (1987);Nelson et al., Nucleosides and Nucleotides, 5(3) 233-241 (1986);Bergstrom, et al., JACS, 111 374-375 (1989); U.S. Pat. Nos. 4,855,225,5,231,191, and 5,449,767, each of which is incorporated herein byreference.

[0102] Preferably, the linkages are acetylenic amido or alkenic amidolinkages, the linkage between the dye and the nucleotide base beingformed by reacting an activated N-hydroxysuccinimide (NHS) ester of thedye with an alkynylamino-, alkynylethoxyamino- oralkenylamino-derivatized base of a nucleotide. More preferably, theresulting linkage is proargyl-1-ethoxyamido(3-(amino)ethoxy-1-propynyl), 3-(carboxy)amino-1-propynyl or3-amino-1-propyn-1-yl.

[0103] Several preferred linkages for linking the dyes of the inventionto a nucleoside base are shown below.

[0104] where R₁ and R₂ taken separately are H, alkyl, a protecting groupor a fluorescent dye.

[0105] The synthesis of alkynylamino-derivatized nucleosides is taughtby Hobbs et al. in European Patent Application No. 87305844.0, and Hobbset al., J. Org. Chem., 54 3420 (1989), which is incorporated herein byreference. Briefly, the alkynylamino-derivatized nucleotides are formedby placing the appropriate halodideoxynucleoside (usually5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides as taughtby Hobbs et al. (cited above)) and Cu(I) in a flask, flushing with argonto remove air, adding dry DMF, followed by addition of an alkynylamine,triethyl-amine and Pd(0). The reaction mixture can be stirred forseveral hours, or until thin layer chromatography indicates consumptionof the halodideoxynucleoside. When an unprotected alkynylamine is used,the alkynylamino-nucleoside can be isolated by concentrating thereaction mixture and chromatographing on silica gel using an elutingsolvent which contains ammonium hydroxide to neutralize the hydrohalidegenerated in the coupling reaction. When a protected alkynylamine isused, methanol/methylene chloride can be added to the reaction mixture,followed by the bicarbonate form of a strongly basic anion exchangeresin. The slurry can then be stirred for about 45 minutes, filtered,and the resin rinsed with additional methanol/methylene chloride. Thecombined filtrates can be concentrated and purified byflash-chromatography on silica gel using a methanol-methylene chloridegradient. The triphosphates are obtained by standard techniques.

[0106] The synthesis of oligonucleotides labeled with an energy transferdye of the present invention can be accomplished using any of a largenumber of known oligonucleotide labeling techniques using knownlinkages, linking groups, and associated complementary functionalities.For example, labeled oligonucleotides may be synthesized enzymatically,e.g., using a DNA polymerase or ligase, e.g., Stryer, Biochemistry,Chapter 24, W. H. Freeman and Company (1981), or by chemical synthesis,e.g., by a phosphoramidite method, a phosphite-triester method, and thelike, e.g., Gait, Oligonucleotide Synthesis, IRL Press (1990). Labelsmay be introduced during enzymatic synthesis utilizing labelednucleoside triphosphate monomers, or introduced during chemicalsynthesis using labeled non-nucleotide or nucleotide phosphoramidites,or may be introduced subsequent to synthesis.

[0107] Generally, if the labeled oligonucleotide is made using enzymaticsynthesis, the following procedure may be used. A template DNA isdenatured and an oligonucleotide primer is annealed to the template DNA.A mixture of deoxynucleoside triphosphates is added to the reactionincluding dGTP, dATP, dCTP, and dTTP where at least a fraction of one ofthe deoxynucleotides is labeled with a dye compound of the invention asdescribed above. Next, a polymerase enzyme is added under conditionswhere the polymerase enzyme is active. A labeled polynucleotide isformed by the incorporation of the labeled deoxynucleotides duringpolymerase strand synthesis. In an alternative enzymatic synthesismethod, two primers are used instead of one, one primer complementary tothe + strand and the other complementary to the − strand of the target,the polymerase is a thermostable polymerase, and the reactiontemperature is cycled between a denaturation temperature and anextension temperature, thereby exponentially synthesizing a labeledcomplement to the target sequence by PCR, e.g., PCR Protocols, Innis etal. eds., Academic Press (1990).

[0108] Generally, if the labeled oligonucleotide is made using achemical synthesis, it is preferred that a phosphoramidite method beused. Phosphoramidite compounds and the phosphoramidite method ofpolynucleotide synthesis are preferred in synthesizing oligonucleotidesbecause of the efficient and rapid coupling and the stability of thestarting materials. The synthesis is performed with the growingoligonucleotide chain attached to a solid support, so that excessreagents, which are in the liquid phase, can be easily removed byfiltration, thereby eliminating the need for purification steps betweencycles.

[0109] In view of the utility of phosphoramidite reagents in labelingnucleosides and oligonucleotides, the present invention also relates tophosphoramidite compounds which include an energy transfer dye of thepresent invention.

[0110] Nucleoside labeling with the dyes can be accomplished using anyof a large number of known nucleoside labeling techniques using knownlinkages, linking groups, and associated complementary functionalities.Preferably, the dyes are covalently linked to the 5-carbon of pyrimidinebases and to the 7-carbon of 7-deazapurine bases. Several suitable baselabeling procedures have been reported that can be used with theinvention, e.g. Gibson et al. Nucleic Acid Res. 15 6455-6467 (1987);Gebeyehu et al. Nucleic Acid Res. 15 4513-4535 (1987); Haralambidis etal. Nucleic Acid Res. 15 4856-4876; Nelson et al. Nucleosides andNucleotides 5 233-241 (1986); Bergstrom et al. J. Am. Chem. Soc.111:374-375 (1989); U.S. Pat. Nos. 4,855,225, 5,231,191, and 5,449,767,each of which is incorporated herein by reference.

[0111] Preferably, the linkages are acetylenic amido or alkenic amidolinkages, the linkage between the dye and nucleotide base being formedby reacting an activated N-hydroxysuccinimide (NHS) ester of the dyewith an alkynylamino, alkynylethoxyamino, or alkenylamino-derivatizedbase of a nucelotide. More preferably, the resulting linkage isproargyl-1-ethoxyamido (3-(amino)ethoxy-1-propynyl),3-(carboxy)amino-1-propynyl or 3-amino-1-propyn-1-yl.

[0112] The synthesis of alkynylamino-derivatized nucleosides is taughtby Hobbs et al. J. Org. Chem. 54:3420 (1989), which is incorporatedherein by reference. Briefly, the alkynylamino-derivatized nucleosidesare formed by placing the appropriate halodideoxynucleoside (usually5-iodopryrimidine and 7-iodo-deazapurine dideoxynucleosides as taught byHobbs et al. as cited above) and Cu(I) in a flask, flushing with argonto remove air, adding dry DMF, followed by addition of an alkynylamine,triethyl-amine and Pd(0). The reaction mixture can be stirred forseveral hours, or until thin layer chromatography indicates consumptionof the halodideoxynucleoside. When an unprotected alkynylamine is used,the alkynylaminonucleoside can be isolated by concentrating the reactionmixture and chromatographing on silica gel using an eluting solventwhich contains ammonium hydroxide to neutralize the hydrohalidegenerated in the coupling reaction. When a protected alkynylamine isused, methanol/methylene chloride can be added to the reaction mixture,followed by the bicarbonate form of a strongly basic anion exchangeresin. The slurry can then be stirred for about 45 minutes, filtered,and the resin rinsed with additional methanol/methylene chloride. Thecombined filtrate can be concentrated and purified byflash-chromatography on silica gel using a methanol-methylene chloridegradient. The triphosphates are obtained by standard techniques.

[0113] Detailed descriptions of the chemistry used to formoligonucleotides by the phosphoramidite method are provided in Carutherset al., U.S. Pat. No. 4,458,066; Caruthers et al., U.S. Pat. No.4,415,732; Caruthers et al., Genetic Engineering, 4 1-17 (1982); UsersManual Model 392 and 394 Polynucleotide Synthesizers, pages 6-1 through6-22, Applied Biosystems, Part No. 901237 (1991), each of which areincorporated by reference in their entirety.

[0114] The following briefly describes the steps of a typicaloligonucleotide synthesis cycle using the phosphoramidite method. First,a solid support including a protected nucleotide monomer is treated withacid, e.g., trichloroacetic acid, to remove a 5′-hydroxyl protectinggroup, freeing the hydroxyl for a subsequent coupling reaction. Anactivated intermediate is then formed by simultaneously adding aprotected phosphoramidite nucleoside monomer and a weak acid, e.g.,tetrazole, to the reaction. The weak acid protonates the nitrogen of thephosphoramidite forming a reactive intermediate. Nucleoside addition iscomplete within 30 s. Next, a capping step is performed which terminatesany polynucleotide chains that did not undergo nucleoside addition.Capping is preferably done with acetic anhydride and 1-methylimidazole.The internucleotide linkage is then converted from the phosphite to themore stable phosphotriester by oxidation using iodine as the preferredoxidizing agent and water as the oxygen donor. After oxidation, thehydroxyl protecting group is removed with a protic acid, e.g.,trichloroacetic acid or dichloroacetic acid, and the cycle is repeateduntil chain elongation is complete. After synthesis, the polynucleotidechain is cleaved from the support using a base, e.g., ammonium hydroxideor t-butyl amine. The cleavage reaction also removes any phosphateprotecting groups, e.g., cyanoethyl. Finally, the protecting groups onthe exocyclic amines of the bases and the hydroxyl protecting groups onthe dyes are removed by treating the polynucleotide solution in base atan elevated temperature, e.g., 55° C.

[0115] Any of the phosphoramidite nucleoside monomers may be dye-labeledphosphoramidites. If the 5′-terminal position of the nucleotide islabeled, a labeled non-nucleotidic phosphoramidite of the invention maybe used during the final condensation step. If an internal position ofthe oligonucleotide is to be labeled, a labeled nucleotidicphosphoramidite of the invention may be used during any of thecondensation steps. Subsequent to their synthesis, oligonucleotides maybe labeled at a number of positions including the 5′-terminus. SeeOligonucleotides and Analogs, Eckstein ed., Chapter 8, IRL Press (1991)and Orgel et al., Nucleic Acids Research 11(18) 6513 (1983); U.S. Pat.No. 5,118,800, each of which are incorporated by reference

[0116] Oligonucleotides may also be labeled on their phosphodiesterbackbone (Oligonucleotides and Analogs, Eckstein ed., Chapter 9) or atthe 3′-terminus (Nelson, Nucleic Acids Research 20(23) 6253-6259, andU.S. Pat. Nos. 5,401,837 and 5,141,813, both patents hereby incorporatedby reference. For a review of oligonucleotide labeling procedures see R.Haugland in Excited States of Biopolymers, Steiner ed., Plenum Press, NY(1983).

[0117] In one preferred post-synthesis chemical labeling method anoligonucleotide is labeled as follows. A dye including a carboxy linkinggroup is converted to the n-hydroxysuccinimide ester by reacting withapproximately 1 equivalent of 1,3-dicyclohexylcarbodiimide andapproximately 3 equivalents of n-hydroxysuccinimide in dry ethyl acetatefor 3 hours at room temperature. The reaction mixture is washed with 5%HCl, dried over magnesium sulfate, filtered, and concentrated to a solidwhich is resuspended in DMSO. The DMSO dye stock is then added in excess(10-20×) to an aminohexyl derivatized oligonucleotide in 0.25 Mbicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6 hours,e.g., U.S. Pat. No. 4,757,141. The dye labeled oligonucleotide isseparated from unreacted dye by passage through a size-exclusionchromatography column eluting with buffer, e.g., 0.1 molar triethylamineacetate (TEAA). The fraction containing the crude labeledoligonucleotide is further purified by reverse phase HPLC employinggradient elution.

[0118] III. Methods Employing Dyes and Reagents of the Present Invention

[0119] The energy transfer dyes and reagents of the present inventionmay be used in a wide variety of methods for detecting the presence of acomponent in a sample by labeling the component in the sample with areagent containing the dye. In particular, the energy transfer dyes andreagents of the present invention are well suited for use in methodswhich combine separation and fluorescent detection techniques,particularly methods requiring the simultaneous detection of multiplespatially-overlapping analytes. For example, the dyes and reagents areparticularly well suited for identifying classes of oligonucleotidesthat have been subjected to a biochemical separation procedure, such aselectrophoresis, where a series of bands or spots of target substanceshaving similar physiochemical properties, e.g. size, conformation,charge, hydrophobicity, or the like, are present in a linear or planararrangement. As used herein, the term “bands” includes any spatialgrouping or aggregation of analytes on the basis of similar or identicalphysiochemical properties. Usually bands arise in the separation ofdye-oligonucleotide conjugates by electrophoresis.

[0120] Classes of oligonucleotides can arise in a variety of contexts.In a preferred category of methods referred to herein as “fragmentanalysis” or “genetic analysis” methods, labeled oligonucleotidefragments are generated through template-directed enzymatic synthesisusing labeled primers or nucleotides, e.g., by ligation orpolymerase-directed primer extension; the fragments are subjected to asize-dependent separation process, e.g., electrophoresis orchromatography; and, the separated fragments are detected subsequent tothe separation, e.g., by laser-induced fluorescence. In a particularlypreferred embodiment, multiple classes of oligonucleotides are separatedsimultaneously and the different classes are distinguished by spectrallyresolvable labels.

[0121] One such fragment analysis method is amplified fragment lengthpolymorphisim detection (AmpFLP) and is based on amplified fragmentlength polymorphisms, i.e., restriction fragment length polymorphismsthat are amplified by PCR. These amplified fragments of varying sizeserve as linked markers for following mutant genes through families. Thecloser the amplified fragment is to the mutant gene on the chromosome,the higher the linkage correlation. Because genes for many geneticdisorders have not been identified, these linkage markers serve to helpevaluate disease risk or paternity. In the AmpFLPs technique, thepolynucleotides may be labeled by using a labeled oligonucleotide PCRprimer, or by utilizing labeled nucleotide triphosphates in the PCR.

[0122] Another fragment analysis method is nick translation. Nicktranslation involves a reaction to replace unlabeled nucleotidetriphosphates in a double-stranded DNA molecule with labeled ones. Free3′-hydroxyl groups are created within the unlabeled DNA by “nicks”caused by deoxyribonuclease I (DNAase I) treatment. DNA polymerase Ithen catalyzes the addition of a labeled nucleotide to the 3′-hydroxylterminus of the nick. At the same time, the 5′ to 3′-exonucleaseactivity of this enzyme eliminates the nucleotide unit from the5′-phosphoryl terminus of the nick. A new nucleotide with a free 3′-OHgroup is incorporated at the position of the original excisednucleotide, and the nick is shifted along by one nucleotide unit in the3′ direction. This 3′ shift will result in the sequential addition ofnew labeled nucleotides to the DNA with the removal of existingunlabeled nucleotides. The nick-translated polynucleotide is thenanalyzed using a separation process, e.g., electrophoresis.

[0123] Another exemplary fragment analysis method is based on variablenumber of tandem repeats, or VNTRs. VNTRs are regions of double-strandedDNA that contain adjacent multiple copies of a particular sequence withthe number of repeating units being variable. Examples of VNTR loci arepYNZ22, pMCT118, and Apo B. A subset of VNTR methods are those methodsbased on the detection of microsatellite repeats, or short tandemrepeats (STRs), i.e., tandem repeats of DNA characterized by a short(2-4 bases) repeated sequence. One of the most abundant interspersedrepetitive DNA families in humans is the (dC-dA)n-(dG-dT)n dinucleotiderepeat family (also called the (CA)n dinucleotide repeat family). Thereare thought to be as many as 50,000 to 100,000 (CA)n repeat regions inthe human genome, typically with 15-30 repeats per block Many of theserepeat regions are polymorphic in length and can therefore serve asuseful genetic markers. Preferably, in VNTR or STR methods, label isintroduced into the polynucleotide fragments by using a dye-labeled PCRprimer.

[0124] Another exemplary fragment analysis method is DNA sequencing. Ingeneral, DNA sequencing involves an extension/termination reaction of anoligonucleotide primer. Included in the reaction mixture aredeoxynucleoside triphosphates (dNTPs) which are used to extend theprimer. Also included in the reaction mixture is at least onedideoxynucleoside triphosphate (ddNTP) which when incorporated onto theextended primer prevents the further extension of the primer. After theextension reaction has been terminated, the different terminationproducts that are formed are separated and analyzed in order todetermine the positioning of the different nucleosides.

[0125] Fluorescent DNA sequencing may generally be divided into twocategories, “dye primer sequencing” and “dye terminator sequencing”. Indye primer sequencing, a fluorescent dye is incorporated onto the primerbeing extended. Four separate extension/termination reactions are thenrun in parallel, each extension reaction containing a differentdideoxynucleoside triphosphate (ddNTP) to terminate the extensionreaction. After termination, the reaction products are separated by gelelectrophoresis and analyzed. See, for example, Ansorge et al., NucleicAcids Res. 15 4593-4602 (1987).

[0126] In one variation of dye primer sequencing, different primers areused in the four separate extension/termination reactions, each primercontaining a different spectrally resolvable dye. After termination, thereaction products from the four extension/termination reactions arepooled, electrophoretically separated, and detected in a single lane.See, for example, Smith et al., Nature 321 674-679 (1986). Thus, in thisvariation of dye primer sequencing, by using primers containing a set ofspectrally resolvable dyes, products from more than oneextension/termination reactions can be simultaneously detected.

[0127] In dye terminator sequencing, a fluorescent dye is attached toeach of the dideoxynucleoside triphosphates. An extension/terminationreaction is then conducted where a primer is extended usingdeoxynucleoside triphosphates until the labeled dideoxynucleosidetriphosphate is incorporated into the extended primer to prevent furtherextension of the primer. Once terminated, the reaction products for eachdideoxynucleoside triphosphate are separated and detected. In oneembodiment, separate extension/termination reactions are conducted foreach of the four dideoxynucleoside triphosphates. In another embodiment,a single extension/termination reaction is conducted which contains thefour dideoxynucleoside triphosphates, each labeled with a different,spectrally resolvable fluorescent dye.

[0128] Thus according to one aspect of the invention, a method isprovided for conducting dye primer sequencing using one or moreoligonucleotide reagents of the present invention. According to thismethod, a mixture of extended labeled primers are formed by hybridizinga nucleic acid sequence with a fluorescently labeled oligonucleotideprimer in the presence of deoxynucleoside triphosphates, at least onedideoxynucleoside triphosphate and a DNA polymerase. The fluorescentlylabeled oligonucleotide primer includes an oligonucleotide sequencecomplementary to a portion of the nucleic acid sequence being sequenced,and an energy transfer fluorescent dye attached to the oligonucleotide.

[0129] According to the method, the DNA polymerase extends the primerwith the deoxynucleoside triphosphates until a dideoxynucleosidetriphosphate is incorporated which terminates extension of the primer.After termination, the mixture of extended primers are separated. Thesequence of the nucleic acid sequence is then determined byfluorescently detecting the mixture of extended primers formed.

[0130] In a further embodiment of this method, four dye primersequencing reactions are run, each primer sequencing reaction includinga different fluorescently labeled oligonucleotide primer and a differentdideoxynucleoside triphosphate (ddATP, ddCTP, ddGTP and ddTTP). Afterthe four dye primer sequencing reactions are run, the resulting mixturesof extended primers may be pooled. The mixture of extended primers maythen be separated, for example by electrophoresis and the fluorescentsignal from each of the four different fluorescently labeledoligonucleotide primers detected in order to determine the sequence ofthe nucleic acid sequence.

[0131] According to a further aspect of the invention, a method isprovided for conducting dye terminator sequencing using one or moredideoxynucleoside triphosphates labeled with an energy transfer dye ofthe present invention. According to this method, a mixture of extendedprimers are formed by hybridizing a nucleic acid sequence with anoligonucleotide primer in the presence of deoxynucleoside triphosphates,at least one fluorescently labeled dideoxynucleotide triphosphate and aDNA polymerase. The fluorescently labeled dideoxynucleotide triphosphateincludes a dideoxynucleoside triphosphate labeled with an energytransfer fluorescent dye of the present invention.

[0132] According to this method, the DNA polymerase extends the primerwith the deoxynucleoside triphosphates until a fluorescently labeleddideoxynucleoside triphosphate is incorporated into the extended primer.After termination, the mixture of extended primers are separated. Thesequence of the nucleic acid sequence is then determined by detectingthe fluorescently labeled dideoxynucleoside attached to the extendedprimer.

[0133] In a further embodiment of this method, the step of forming amixture of extended primers includes hybridizing the nucleic acidsequence with four different fluorescently labeled dideoxynucleosidetriphosphates, i.e., a fluorescently labeled dideoxycytosinetriphosphate, a fluorescently labeled dideoxyadenosine triphosphate, afluorescently labeled dideoxyguanosine triphosphate, and a fluorescentlylabeled dideoxythymidine triphosphate.

[0134] In each of the above-described fragment analysis methods, thelabeled oligonucleotides are preferably separated by electrophoreticprocedures, e.g. Gould and Matthews, cited above; Rickwood and Hames,Eds., Gel Electrophoresis of Nucleic Acids: A Practical Approach, (IRLPress Limited, London, 1981); or Osterman, Methods of Protein andNucleic Acid Research, Vol. 1 Springer-Verlag, Berlin, 1984). Preferablythe type of electrophoretic matrix is crosslinked or uncrosslinkedpolyacrylamide having a concentration (weight to volume) of betweenabout 2-20 weight percent. More preferably, the polyacrylamideconcentration is between about 4-8 percent. Preferably in the context ofDNA sequencing in particular, the electrophoresis matrix includes astrand separating, or denaturing, agent, e.g., urea, formamide, and thelike. Detailed procedures for constructing such matrices are given byManiatis et al., “Fractionation of Low Molecular Weight DNA and RNA inPolyacrylamide Gels Containing 98% Formamide or 7M Urea,” in Methods inEnzymology, 65 299-305 (1980); Maniatis et al., “Chain LengthDetermination of Small Double- and Single-Stranded DNA Molecules byPolyacrylamide Gel Electrophoresis,” Biochemistry, 14 3787-3794 (1975);Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, New York, 1982), pgs. 179-185; and ABI PRISM™ 377 DNASequencer User∓s Manual, Rev. A, January 1995, Chapter 2 (p/n 903433,The Perkin-Elmer Corporation, Foster City, Calif.), each of which areincorporated by reference. The optimal polymer concentration, pH,temperature, concentration of denaturing agent, etc. employed in aparticular separation depends on many factors, including the size rangeof the nucleic acids to be separated, their base compositions, whetherthey are single stranded or double stranded, and the nature of theclasses for which information is sought by electrophoresis. Accordinglyapplication of the invention may require standard preliminary testing tooptimize conditions for particular separations. By way of example,oligonucleotides having sizes in the range of between about 20-300 baseshave been separated and detected in accordance with the invention in thefollowing matrix: 6 percent polyacrylamide made from 19 parts to 1 partacrylamide to bis-acrylamide, formed in a Tris-borate EDTA buffer at pH8.3.

[0135] After electrophoretic separation, the dye-oligonucleotideconjugates are detected by measuring the fluorescence emission from thedye labeled polynucleotides. To perform such detection, the labeledpolynucleotides are illuminated by standard light sources, e.g. highintensity mercury vapor lamps, lasers, or the like. Previously,fluorescein and rhodamine—based dyes and fluorescein-linked energytransfer dyes have been used which are excited at a wavelength between488 and 550 nm. However, the donor dyes used in the energy transfer dyesof the present invention typically have absorption maxima below 450 nmand thus may be excited at shorter wavelengths, preferably between 250and 450 nm.

[0136] IV. Detection Methods Using Shorter Wavelength Light Sources

[0137] The present invention also relates to detection methods, such asthe detection methods described above in Section III, in which a shorterwavelength light source is used, preferably a light source emittinglight between 250 and 450 nm. As noted above, several of the energytransfer dyes of the present invention have the feature of having adonor dye with an emission maxima between about 250 and 450 nm and anacceptor dye which has an emission maxima at a wavelength greater thanabout 500 nm. As a result, these dyes enable these shorter wavelengthlight sources to be used. Accordingly, the present invention relates tomethods for using these shorter wavelength light sources. It is notedthat the use of these shorter wavelength light sources in detectionmethods, such as the ones described in Section III, is not intended tobe limited to the energy transfer dyes of the present invention butrather are intended to encompass the use of any energy transfer dyewhich can be excited using light having a wavelength between 250 and 450nm

[0138] V. Kits Incorporating the Energy Transfer Dyes

[0139] The present invention also relates to kits having combinations ofenergy transfer dyes and/or reagents. In one embodiment, the kitincludes at least two spectrally resolvable energy transfer dyesaccording to the present invention. In this kit, the energy transferdyes preferably include the same donor dye so that a single light sourceis needed to excite the dyes.

[0140] In another embodiment, the kit includes dideoxycytosinetriphosphate, dideoxyadenosine triphosphate, dideoxyguanosinetriphosphate, and dideoxythymidine triphosphate, each dideoxynucleotidetriphosphate labeled with an energy transfer dye according to thepresent invention. In one embodiment, each energy transfer dye isspectrally resolvable from the other energy transfer dyes attached tothe other dideoxynucleotide triphosphates. In this kit, the energytransfer dyes preferably include the same first xanthene dye.

[0141] In yet another embodiment, the kit includes at least twooligonucleotides, each oligonucleotide including an energy transfer dyeaccording to the present invention. In one embodiment, eacholigonucleotide contains an energy transfer dye which is spectrallyresolvable from the energy transfer dyes attached to the otheroligonucleotides. In another embodiment, the kit includes at least fouroligonucleotides which each contain a spectrally resolvable energytransfer dye.

[0142] The energy transfer dyes and their use in DNA sequencing isillustrated by the following examples. Further objectives and advantagesother than those set forth above become apparent from the examples.

EXAMPLES

[0143] 1. Method of Synthesis of DYE104

[0144] A solution of 5CF-B (8 mg in 0.45 mL dimethylformamide (DMF), 20μL) was added to a solution of the succimidyl ester of coumarin DYE114(20 μL of a 5 mg/200 μL DMF solution). Diisopropylethylamine (5 μL) wasadded. After 5 min, 200 μL of 5% HCl was added. The mixture wascentrifuged. The solid was dissolved in bicarbonate solution andpurified by reverse-phase HPLC. The synthesis scheme of DYE104 isillustrated in FIG. 9.

[0145] 2. Method of Synthesis of DYE106

[0146] A solution of 5CF-B (8 mg in 0.45 mL dimethylformamide (DMF), 20μL) was added to a solution of the succimidyl ester of coumarin DYE116(20 μL of a 5 mg/200 μL DMF solution). Diisopropylethylamine (5 μL) wasadded. After 5 min, 200 μL of 5% HCl was added. The mixture wascentrifuged. The solid was dissolved in bicarbonate solution andpurified by reverse-phase HPLC. The synthesis scheme of DYE106 isillustrated in FIG. 10.

[0147] 2. Method of Synthesis of DYE108

[0148] A solution of 5CF-B (8 mg in 0.45 mL dimethylformamide (DMF), 20μL) was added to a solution (20 μL of a 5 mg/200 μL DMF solution) ofDYE110, trisulfopyrene acetyl azide (or Cascade Blue acetyl azide,Molecular Probes). Diisopropylethylamine (5 μL) was added. After 5 min,200 μL of 5% HCl was added. The mixture was centrifuged. The solid wasdissolved in bicarbonate solution and purified by reverse-phase HPLC.The synthesis scheme of DYE108 is illustrated in FIG. 11.

[0149] 3. Comparison of Fluorescence Emission Spectra of5CF-B-Conjugates

[0150] The following example compares the fluorescence emission spectraof a series of energy transfer dyes according to the present invention.Dye solutions of 5CF-B, DYE102, DYE104, DYE106, and DYE108 were measuredin Tris-EDTA.

[0151] The structures of these dyes are illustrated in FIG. 1. FIG. 12provides a graph of the relative fluorescence emission of each of thesedyes when excited at 365 nm. FIG. 12 also show the emission maxima ofthe individual dye components. As shown in FIG. 12, the emissions of thedonor dyes do not overlap with the absorbance of the acceptor dye. Thebest conjugate, DYE108, is more than 10-fold brighter than 5CF-B alone.

[0152] Table 1 shows the relative spectral data and relative quantumyields of 5CF-B conjugates. As can be seen from Table 1, the quantumyields are high and the energy transfer is practically quantitative, asobserved by the lack of emission of the donor dyes. Coumarin based dyesDYE104 and DYE102 and pyrene based dye DYE108 display high quantumyields indicating that the acceptor is able to absorb substantially allof the energy emitted by the donor dye. In contrast, the DYE106(coumarin) displays poor quantum yield and inefficient energy transfer.TABLE 1 E_(X)/E_(M) Maxima of Quantum Yield of Conjugate 5CF-B conjugateIndividual Dyes (nm) Relative to 5CF-B 5CF-B 495/523 1.00 DYE106 376/4680.17 DYE104 328/386 0.93 DYE108 396/410 0.91 DYE102 362/459 0.87

[0153] 4. Method of Synthesis of Pyrenetrisulfonate-Rhodamine Dye(DYE120)

[0154] D-Rox succinimidyl ester (3 mg), 1,4-cyclohexanediamine (7 mg),DMF (100 μL) and diisopropylethylamine (10 μL) were combined. After 5min ethyl ether was added. The mixture was centrifuged and decanted. Theresidue was dissolved in methanol and an aliquot was purified byreverse-phase HPLC to separate the d-Rox-acid from the thed-Rox-cyclohexanediamine adduct. The purified adduct was concentrated todryness and dissolved in 10 μL DMF.

[0155] A solution of DYE110, Cascade Blue acetyl azide, was made(Molecular Probes, 8 mg/100 μL DMF). To 5 μL of the Dye110 solution wasadded the d-Rox-cyclohexanediamine adduct and 2 μL diisopropylamine. Themixture was purified by reverse-phase HPLC. The synthesis scheme ofpyrenetrisulfonate-d-Rox dye (DYE120) is illustrated in FIG. 13.

[0156] Normalized excitation and emission spectra of thepyrenetrisulfonate-d-Rox adduct (DYE120) are shown in FIG. 14. Verylittle pyrenetrisulfonate emission (410 nm) was observed. The excitationspectra showed a peak at 400 nm that was 50% of the maximum peak at 600nm.

[0157] While the present invention is disclosed by reference to thepreferred embodiments and examples detailed above, it is to beunderstood that these examples are intended in an illustrative ratherthan limiting sense, as it is contemplated that modifications willreadily occur to those skilled in the art, which modifications will bewithin the spirit of the invention and the scope of the appended claims.With regard to all of the molecular structures provided herein, it isintended that these molecular structures encompass not only the exactelectronic structure presented, but also include all resonant structuresand protonation states thereof.

What is claimed is:
 1. An energy transfer dye comprising: a donor dyehaving an absorption maxima at a wavelength between about 250 to 450 nm;and an acceptor dye capable of absorbing excitation energy emitted bythe donor dye and emitting light in response, the acceptor dye having anemission maxima greater than about 500 nm.
 2. The energy transfer dyeaccording to claim 1 wherein the donor dye has an absorption maxima at awavelength between about 300 and 450 nm.
 3. The energy transfer dyeaccording to claim 1 wherein the donor dye has an absorption maxima at awavelength between about 350 and 400 nm.
 4. The energy transfer dyeaccording to claim 1 wherein the acceptor dye has an emission maxima ata wavelength greater than about 550 nm.
 5. The energy transfer dyeaccording to claim 1 wherein the acceptor dye has an emission maxima ata wavelength between about 500 and 700 nm.
 6. The energy transfer dyeaccording to claim 1 wherein the acceptor dye has an emission maxima ata wavelength at least about 150 nm greater than the absorption maxima ofthe donor dye.
 7. The energy transfer dye according to claim 1 whereinthe acceptor dye is a member of a class of dyes selected from the groupconsisting of fluorescein, rhodamine, asymmetric benzoxanthene,xanthene, cyanine, phthalocyanine and squaraine dyes.
 8. The energytransfer dye according to claim 1 wherein the acceptor dye is selectedfrom the group consisting of 4,7-dichlorofluorescein dyes, asymmetricbenzoxanthene dyes, rhodamine, 4,7-dichlororhodamine dyes,carboxyrhodamines, N,N,N′,N′-tetramethyl carboxyrhodamines, carboxyR110, carboxy R6G, carboxy-X-rhodamines and Cy5.
 9. The energy transferdye according to claim 1 wherein the acceptor dye is selected from thegroup consisting of R110, RG6, TAMRA and ROX.
 10. The energy transferdye according to claim 1 further comprising a linker attaching the donordye to the acceptor dye.
 11. The energy transfer dye according to claim10 wherein the linker linking the donor dye to the acceptor dye is suchthat the acceptor dye absorbs substantially all of the excitation energyemitted by the donor dye, the linker including a functional groupselected from the group consisting of an alkene, diene, alkyne, a fiveand six membered ring having at least one unsaturated bond or a fusedring structure.
 12. The energy transfer dye according to claim 11wherein the linker functional group is a five or six membered ringselected from the group consisting of cyclopentene, cyclohexene,cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole,isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine,pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran,thionaphthene, indole and naphthalene.
 13. An energy transfer dyecomprising: a donor dye which is a member of a class of dyes having acoumarin or pyrene ring structure; and an acceptor dye capable ofabsorbing excitation energy emitted by the donor dye and emitting lightin response, the acceptor dye having an emission maxima greater thanabout 500 nm.
 14. The energy transfer dye according to claim 13 whereinthe donor dye has an absorption maxima at a wavelength between about 250and 450 nm.
 15. The energy transfer dye according to claim 13 whereinthe donor dye has an absorption maxima at a wavelength between about 300and 450 nm.
 16. The energy transfer dye according to claim 13 whereinthe donor dye has an absorption maxima at a wavelength between about 350and 400 nm.
 17. The energy transfer dye according to claim 13 whereinthe acceptor dye has an emission maxima at a wavelength greater thanabout 550 nm.
 18. The energy transfer dye according to claim 13 whereinthe acceptor dye has an emission maxima at a wavelength between about500 and 700 nm.
 19. The energy transfer dye according to claim 13wherein the acceptor dye has an emission maxima at a wavelength at leastabout 150 nm greater than the absorption maxima of the donor dye. 20.The energy transfer dye according to claim 13 wherein the donor dye isselected from the group consisting of coumarin, pyrene and pyrenesulfonate.
 21. The energy transfer dye according to claim 13 wherein theacceptor dye is a member of a class of dyes selected from the groupconsisting of fluorescein, rhodamine, asymmetric benzoxanthene,xanthene, cyanine, phthalocyanine and squaraine dyes.
 22. The energytransfer dye according to claim 13 wherein the acceptor dye is selectedfrom the group consisting of 4,7-dichlorofluorescein dyes, asymmetricbenzoxanthene dyes, rhodamine, 4,7-dichlororhodamine dyes,carboxyrhodamines, N,N,N′,N′-tetramethyl carboxyrhodamines, carboxyR110, carboxy R6G, carboxy-X-rhodamines and Cy5.
 23. The energy transferdye according to claim 13 wherein the acceptor dye is selected from thegroup consisting of R110, RG6, TAMRA and ROX.
 24. The energy transferdye according to claim 13 further comprising a linker attaching thedonor dye to the acceptor dye.
 25. The energy transfer dye according toclaim 24 wherein the linker linking the donor dye to the acceptor dye issuch that the acceptor dye absorbs substantially all of the excitationenergy emitted by the donor dye, the linker including a functional groupselected from the group consisting of an alkene, diene, alkyne, a fiveand six membered ring having at least one unsaturated bond or a fusedring structure.
 26. The energy transfer dye according to claim 25wherein the linker functional group is a five or six membered ringselected from the group consisting of cyclopentene, cyclohexene,cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole,isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine,pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran,thionaphthene, indole and naphthalene.
 27. An energy transfer dyeselected from the group consisting of DYE102, DYE104, DYE106, DYE108,DYE118 and DYE120.
 28. A fluorescently labeled reagent comprising: areagent selected from the group consisting of a nucleoside, nucleosidemonophosphate, nucleoside diphosphate, nucleoside triphosphate,oligonucleotide and oligonucleotide analog, modified to be linked to anenergy transfer fluorescent dye; and an energy transfer fluorescent dyeattached to the reagent, the energy transfer fluorescent dye including adonor dye having an absorption maxima at a wavelength between about 250to 450 nm; and an acceptor dye capable of absorbing excitation energyemitted by the donor dye and emitting light in response, the acceptordye having an emission maxima greater than about 500 nm.
 29. The energytransfer dye according to claim 28 wherein the donor dye has anabsorption maxima at a wavelength between about 300 and 450 nm.
 30. Thefluorescently labeled reagent according to claim 28 wherein the donordye has an absorption maxima at a wavelength between about 350 and 400nm.
 31. The fluorescently labeled reagent according to claim 28 whereinthe acceptor dye has an emission maxima at a wavelength greater thanabout 550 nm.
 32. The fluorescently labeled reagent according to claim28 wherein the acceptor dye has an emission maxima at a wavelengthbetween about 500 and 700 nm.
 33. The fluorescently labeled reagentaccording to claim 28 wherein the acceptor dye has an emission maxima ata wavelength at least about 150 nm greater than the absorption maxima ofthe donor dye.
 34. The fluorescently labeled reagent according to claim28 wherein the acceptor dye is is a member of a class of dyes selectedfrom the group consisting of fluorescein, rhodamine, asymmetricbenzoxanthene, xanthene, cyanine, phthalocyanine and squaraine dyes. 35.The fluorescently labeled reagent according to claim 28 wherein theacceptor dye is selected from the group consisting of4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine,4,7-dichlororhodamine dyes, carboxyrhodamines, N,N,N′,N′-tetramethylcarboxyrhodamines, carboxy R110, carboxy R6G, carboxy-X-rhodamines andCy5.
 36. The fluorescently labeled reagent according to claim 28 whereinthe acceptor dye is selected from the group consisting of R110, RG6,TAMRA and ROX.
 37. The fluorescently labeled reagent according to claim28 further comprising a linker attaching the donor dye to the acceptordye.
 38. The fluorescently labeled reagent according to claim 37 whereinthe linker linking the donor dye to the acceptor dye is such that theacceptor dye absorbs substantially all of the excitation energy emittedby the donor dye, the linker including a functional group selected fromthe group consisting of an alkene, diene, alkyne, a five and sixmembered ring having at least one unsaturated bond or a fused ringstructure.
 39. The fluorescently labeled reagent according to claim 38wherein the linker functional group is a five or six membered ringselected from the group consisting of cyclopentene, cyclohexene,cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole,isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine,pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran,thionaphthene, indole and naphthalene.
 40. The fluorescently labeledreagent according to claim 28 wherein the reagent is selected from thegroup consisting of deoxynucleoside, deoxynucleoside monophosphate,deoxynucleoside diphosphate and deoxynucleoside triphosphate.
 41. Thefluorescently labeled reagent according to claim 40 wherein thedeoxynucleotides are selected from the group consisting ofdeoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine. 42.The fluorescently labeled reagent according to claim 28 wherein thereagent is selected from the group consisting of dideoxynucleoside,dideoxynucleoside monophosphate, dideoxynucleoside diphosphate anddideoxynucleoside triphosphate.
 43. The fluorescently labeled reagentaccording to claim 42 wherein the dideoxynucleotides are selected fromthe group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine,and deoxythymidine.
 44. The fluorescently labeled reagent according toclaim 28 wherein the reagent is an oligonucleotide.
 45. Thefluorescently labeled reagent according to claim 44 wherein theoligonucleotide has a 3′ end which is extendable by using a polymerase.46. A fluorescently labeled reagent comprising: a reagent selected fromthe group consisting of a nucleoside, nucleoside monophosphate,nucleoside diphosphate, nucleoside triphosphate, oligonucleotide andoligonucleotide analog, modified to be linked to an energy transferfluorescent dye; and an energy transfer fluorescent dye attached to thereagent, the energy transfer fluorescent dye including a donor dye whichis a member of a class of dyes having a coumarin or pyrene ringstructure; and an acceptor dye capable of absorbing excitation energyemitted by the donor dye and emitting light in response, the acceptordye having an emission maxima greater than about 500 nm.
 47. Thefluorescently labeled reagent according to claim 46 wherein the donordye has an absorption maxima at a wavelength between about 250 and 450nm.
 48. The fluorescently labeled reagent according to claim 46 whereinthe donor dye has an absorption maxima at a wavelength between about 300and 450 nm.
 49. The fluorescently labeled reagent according to claim 46wherein the donor dye has an absorption maxima at a wavelength betweenabout 350 and 400 nm.
 50. The fluorescently labeled reagent according toclaim 46 wherein the acceptor dye has an emission maxima at a wavelengthgreater than about 550 nm.
 51. The fluorescently labeled reagentaccording to claim 46 wherein the acceptor dye has an emission maxima ata wavelength between about 500 and 700 nm.
 52. The fluorescently labeledreagent according to claim 46 wherein the acceptor dye has an emissionmaxima at a wavelength at least about 150 nm greater than the absorptionmaxima of the donor dye.
 53. The fluorescently labeled reagent accordingto claim 46 wherein the donor dye is selected from the group consistingof coumarin, pyrene and pyrene sulfonate.
 54. The fluorescently labeledreagent according to claim 46 wherein the acceptor dye is a member of aclass of dyes selected from the group consisting of fluorescein,rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanineand squaraine dyes.
 55. The fluorescently labeled reagent according toclaim 46 wherein the acceptor dye is selected from the group consistingof 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes,rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines,N,N,N′,N′-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G,carboxy-X-rhodamines and Cy5.
 56. The fluorescently labeled reagentaccording to claim 46 wherein the acceptor dye is selected from thegroup consisting of R110, RG6, TAMRA and ROX.
 57. The fluorescentlylabeled reagent according to claim 46 further comprising a linkerattaching the donor dye to the acceptor dye.
 58. The fluorescentlylabeled reagent according to claim 57 wherein the linker linking thedonor dye to the acceptor dye is such that the acceptor dye absorbssubstantially all of the excitation energy emitted by the donor dye, thelinker including a functional group selected from the group consistingof an alkene, diene, alkyne, a five and six membered ring having atleast one unsaturated bond or a fused ring structure.
 59. Thefluorescently labeled reagent according to claim 58 wherein the linkerfunctional group is a five or six membered ring selected from the groupconsisting of cyclopentene, cyclohexene, cyclopentadiene,cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole,pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine,pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indoleand naphthalene.
 60. The fluorescently labeled reagent according toclaim 46 wherein the reagent is selected from the group consisting ofdeoxynucleoside, deoxynucleoside monophosphate, deoxynucleosidediphosphate and deoxynucleoside triphosphate.
 61. The fluorescentlylabeled reagent according to claim 60 wherein the deoxynucleotides areselected from the group consisting of deoxycytosine, deoxyadenosine,deoxyguanosine, and deoxythymidine.
 62. The fluorescently labeledreagent according to claim 46 wherein the reagent is selected from thegroup consisting of dideoxynucleoside, dideoxynucleoside monophosphate,dideoxynucleoside diphosphate and dideoxynucleoside triphosphate. 63.The fluorescently labeled reagent according to claim 62 wherein thedideoxynucleotides are selected from the group consisting ofdeoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine. 64.The fluorescently labeled reagent according to claim 46 wherein thereagent is an oligonucleotide.
 65. The fluorescently labeled reagentaccording to claim 64 wherein the oligonucleotide has a 3′ end which isextendable by using a polymerase.
 66. A fluorescently labeled reagentcomprising: a reagent selected from the group consisting of anucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleosidetriphosphate, oligonucleotide and oligonucleotide analog, modified to belinked to an energy transfer fluorescent dye; and an energy transferfluorescent dye attached to the reagent, the energy transfer fluorescentdye being selected from the group consisting of DYE102, DYE104, DYE106,DYE108, DYE118 and DYE120.
 67. A method for sequencing a nucleic acidsequence comprising: forming a mixture of extended labeled primers byhybridizing a nucleic acid sequence with a fluorescently labeledoligonucleotide primer in the presence of deoxynucleoside triphosphates,at least one dideoxynucleoside triphosphate and a DNA polymerase, theDNA polymerase extending the primer with the deoxynucleosidetriphosphates until a dideoxynucleoside triphosphate is incorporatedwhich terminates extension of the primer; separating the mixture ofextended primers; and determining the sequence of the nucleic acidsequence by fluorescently measuring the mixture of extended primersformed; the fluorescently labeled oligonucleotide primer including anoligonucleotide sequence complementary to a portion of the nucleic acidsequence being sequenced and having a 3′ end extendable by a polymerase,and an energy transfer fluorescent dye attached to the oligonucleotide,the energy transfer fluorescent dye including a donor dye having anabsorption maxima at a wavelength between about 250 to 450 nm; and anacceptor dye capable of absorbing excitation energy emitted by the donordye and emitting light in response, the acceptor dye having an emissionmaxima greater than about 500 nm.
 68. A method for sequencing a nucleicacid sequence comprising: forming a mixture of extended labeled primersby hybridizing a nucleic acid sequence with a fluorescently labeledoligonucleotide primer in the presence of deoxynucleoside triphosphates,at least one dideoxynucleoside triphosphate and a DNA polymerase, theDNA polymerase extending the primer with the deoxynucleosidetriphosphates until a dideoxynucleoside triphosphate is incorporatedwhich terminates extension of the primer; separating the mixture ofextended primers; and determining the sequence of the nucleic acidsequence by fluorescently measuring the mixture of extended primersformed, the fluorescently labeled oligonucleotide primer including anoligonucleotide sequence complementary to a portion of the nucleic acidsequence being sequenced and having a 3′ end extendable by a polymerase,and an energy transfer fluorescent dye attached to the oligonucleotide,the energy transfer fluorescent dye including a donor dye which is amember of a class of dyes having a coumarin or pyrene ring structure;and an acceptor dye capable of absorbing excitation energy emitted bythe donor dye and emitting light in response, the acceptor dye having anemission maxima greater than about 500 nm.
 69. A method for sequencing anucleic acid sequence comprising: forming a mixture of extended primersby hybridizing a nucleic acid sequence with a primer in the presence ofdeoxynucleoside triphosphates, at least one fluorescently labeleddideoxynucleoside triphosphate and a DNA polymerase, the DNA polymeraseextending the primer with the deoxynucleoside triphosphates until afluorescently labeled dideoxynucleoside triphosphate is incorporatedonto the extended primer which terminates extension of the primer;separating the mixture of extended primers; and determining the sequenceof the nucleic acid sequence by detecting the fluorescently labeleddideoxynucleotide attached to the separated mixture of extended primers;the fluorescently labeled dideoxynucleoside triphosphate including adideoxynucleoside triphosphate, and an energy transfer fluorescent dyeattached to the dideoxynucleoside triphosphate, the energy transfer dyeincluding a donor dye having an absorption maxima at a wavelengthbetween about 250 to 450 nm; and an acceptor dye capable of absorbingexcitation energy emitted by the donor dye and emitting light inresponse, the acceptor dye having an emission maxima greater than about500 nm.
 70. A method for sequencing a nucleic acid sequence comprising:forming a mixture of extended primers by hybridizing a nucleic acidsequence with a primer in the presence of deoxynucleoside triphosphates,at least one fluorescently labeled dideoxynucleoside triphosphate and aDNA polymerase, the DNA polymerase extending the primer with thedeoxynucleoside triphosphates until a fluorescently labeleddideoxynucleoside triphosphate is incorporated onto the extended primerwhich terminates extension of the primer; separating the mixture ofextended primers; and determining the sequence of the nucleic acidsequence by detecting the fluorescently labeled dideoxynucleotideattached to the separated mixture of extended primers; the fluorescentlylabeled dideoxynucleoside triphosphate including a dideoxynucleosidetriphosphate, and an energy transfer fluorescent dye attached to thedideoxynucleoside triphosphate, the energy transfer dye including adonor dye which is a member of a class of dyes having a coumarin orpyrene ring structure; and an acceptor dye capable of absorbingexcitation energy emitted by the donor dye and emitting light inresponse, the acceptor dye having an emission maxima greater than about500 nm.